Formation of 2-keto-3-deoxy aldonic acids by cell-free extracts of Aspergillus ustus Aii M. Elshafei and Osama M. Abdel-Fatah Laboratory of Microbial Chemistry, National Research Centre, Dokki, Cairo, Egypt Different aldolase activities were demonstrated in cell-free extracts of Aspergillus ustus grown on o-ghlcose or o-gluconate as the only source of carbon. Pyruvic, oxalacetic, or glycolic acids were used as substrates for enzymatic condensation with different aldehydes. 2-Keto-3-deoxy arabonic acid (KDA) or 2-keto-3-deoxy gluconic acid (KDG) was the product of such condensation. No activity could be detected when glycolic acid, formaldehyde, or acetaldehyde was used as substrate. Maximum pH and temperature were found to be 7.5 and 50°C for KDA aldolase, and 7.0 and 50°C for KDG adolase. Decreased activity was obtained when o-glucose replaced o-gluconate in the fermentation medium. Thermal stability behavior of KDG aldolase indicates that temperature has a stimulating effect on enzyme activity at 50°C. HgCI2, para-mercurychlorobenzoate, ZnS04, and CuSO~ were potent inhibitors of this enzyme. The Km values were determined for pyruvate and glyceraldehyde and were found to be 4.1 x 10-~ and 7.1 x lO-3M, respectively. Keywords: Aldehydes; ketonic acids; 2-keto-3-deoxy-gluconate aldolase; Aspergillus ustus Introduction There are few reported studies of the presence of different aldolases in bacteria and fungi. Dahms and Anderson t proved the presence of an aldolase that catalyses the cleavage of 2-keto-3-deoxy-L-arabonate to pyruvate and glycolaldehyde in extracts of a Pseu- domonad MSU-I grown on L-arabinose. In 1972 the same authors demonstrated the presence of an al- dolase that catalyses the cleavage of 2-keto- 3-deoxy-D-fuconate to D-lactaldehyde and pyruvate. Tracer experiments proved that the carbonyl group of pyruvate is derived from Ct of 2-keto-3-deoxy-o-fuco- nate. 2 Andreesen and Gottschalk 3 found that cell-free extracts of Clostridium aceticum, when grown anaero- bically, contain gluconate dehydratase, 2-keto-3- deoxyglucokinase, and 2-keto-3-deoxy-6-phosphoglu- conic acid aldolase. Elzainy and Allam 4 reported the presence of 2-keto- 3-deoxy-o-gluconate (KDG) aldolase in Aspergillus niger grown on D-gluconate. KDG aldolase of A. niger was then purified and some of its properties were Address reprint requests to Dr. Elshafei at the Laboratory of Microbial Chemistry, National Research Centre, Dokki, Cairo, Egypt Received 3 March 1988; revised 1 June 1988 studied. 5 Elshafei 6 and Elzainy et al. 7 have indicated that cell-free extracts of L-arabonate-grown mycelia of A. niger catalysed the cleavage of 2-keto-3-deoxy-L- arabonate (KDA) into pyruvate and glycolaldehyde, as well as the condensation of the latter two products into KDA. Recently Elshafei 8 reported the presence of an aldolase that catalyses the condensation of glyceralde- hyde and pyruvate to KDG in A. niger, as a part of a new nonphosphorylative metabolic pathway for D-glu- cose degradation by cell-free extracts of the same strain grown on D-glucose as sole source of carbon. The present paper represents a study of different aldolases in cell-free extracts of Aspergillus ustus using different ketonic acids and aldehydes as sub- strates. Some properties of KDG aldolase were studied. Materials and methods Organism and media Aspergillus ustus was obtained from the culture col- lection of Cairo Mircen Ain-Shams University. The organism was grown on Czapek-Dox medium at pH 6.0 with o-glucose or D-gluconate (2%) as sole source of carbon, In some experiments 2.0 g 1 -j malt extract were incorporated to the medium to stimulate good fungal growth. © 1989 Butterworth Publishers Enzyme Microb. Technol., 1989, vol. 11, June 367